The long term objective of this project is to understand the biogenetic mechanisms responsible for the subcellular distribution of specific proteins in membranes and organelles. We will focus on molecular aspects of the mechanisms that determine the disposition with respect to the phospholipid bilayer of membrane polypeptides synthesized in the ER. We will also study the sorting processes that are involved in the retention of proteins in the ER or determine their transfer to the Golgi apparatus, and from there, to lysosomes or the plasma membrane. Cytochrome P-450, and integral membrane protein of the ER which is cotranslationally inserted in the membrane but remains largely exposed on its cytoplasmic surface, has been chosen as a model protein for some of this work. The essential structural features that enable the uncleaved insertion signal in P-450 to halt transfer of distal sequences and to anchor the mature protein in the phospholipid bilayer, will be identified by examining the membrane insertion of chimeric and altered polypeptides generated in in vitro transcription-translation systems programmed with genetically engineered templates. These studies will also provide insights into the features of other uncleaved signals, which do not have halt transfer function but either pass through the membrane or remain permanently anchored to it. The studies concerning the posttranslational sorting of proteins inserted in the ER, include: 1) The isolation and biochemical characterization of the transitional cisternae that provide the site of exit for proteins delivered from the ER to the Golgi apparatus. for these studies, cells that synthesize an abnormal chimeric protein that accumulates in this organelle and leads to its overdevelopment will be employed. Attempts will be made to identify receptors of carriers that, complexed to the chimeric protein, may accumulate within these overdeveloped cisternae. 2) The identification of structural features in beta- glucuronidase responsible for the phosphorylation of the mannose residues that determine the sequestration of this protein in lysosomes. 3) an elucidation of the pathway followed by a lysosomal membrane protein to lysosomes and the identification of the sorting signal that address this protein to its destination. 4) An identification of the features that lead to the retention of cytochrome P-450 within the ER membrane and of a microsomal esterase within the ER lumen.